Process for producing 1,4-dihydroxy-2-naphthoic acid

ABSTRACT

A composition containing 1,4-dihydroxy-2-naphthoic acid at a high concentration is obtained by intracellularly and extracellularly producing 1,4-dihydroxy-2-naphthoic acid using a bacterium belonging to the genus  Propionibacterium  and collecting it. This composition is efficacious in improving intestinal flora, alleviating abdominal ailments in association with the intake of milk, and preventing metabolic bone diseases.

TECHNICAL FIELD

The present invention relates to an industrial production process for1,4-dihydroxy-2-naphthoic acid (or 1,4-dihydroxy-2-naphthalenecarboxylic acid) (hereinafter may be referred to simply as “DHNA”); to adrug containing the compound; and to food and beverage which contain thecompound and thus are useful for improving intestinal flora, alleviatingabdominal ailments caused by lactose intolerance, and for preventing ortreating metabolic bone diseases.

BACKGROUND ART

Comparative studies on the intestinal flora found in breast-fed infantsand that in bottle-fed infants have suggested that bifidobacteria areuseful for improving human health. As has been confirmed, the amount ofbifidobacteria in the body is significantly reduced by gastrointestinaltract diseases or similar diseases, or aging, and promotion ofproliferation of intestinal bifidobacteria is effective for preventingcarcinogenesis, intestinal putrefaction, infectious diseases, and otherhealth problems. Therefore, selective proliferation of intestinalbifidobacteria is considered very important for maintaining health andpreventing and treating various diseases, including life-style-relateddiseases.

Conventionally, several substances capable of promoting proliferation ofuseful bifidobacteria—such a substance is called a “Bifidus factor”—havebeen studied and reported. Examples of the substances includeN-acetylglucosamine, which is found in breast milk (Proc. Soc. Exp.Biol. Med., 90, 219 (1955)); peptide-related substances (Am. J. Clin.Nutr., 32, 1428 (1974) and Agric. Biol. Chem., 48, 2159 (1984)); carrotextracts (Journal of Japan Society for Bioscience, Biotechnology, andAgrochemistry, 55, 499 (1981) and Chem. Pharm. Bull., (Tokyo) 14, 1191(1966)); and sugar-related substances (Proceedings of Tohoku FukushiUniversity, 10, 313 (1986)).

However, preparation of any of these substances capable of promotingproliferation of bifidobacteria requires an intricate process, and theeffect of such substances in terms of selective proliferation of merelybifidobacteria needs to be improved.

In view of the foregoing, the present inventors have performed extensivestudies on compounds capable of selectively promoting proliferation ofbifidobacteria, and as a result have found that certain types ofnaphthoquinone derivatives and naphthalene derivatives exhibit strongactivity in promoting proliferation of various bifidobacteria (e.g.,Bifidobacterium longum, B. breve, B. adolescentis, B. bifidum, B.infantis, B. animalis, and B. pseudolongum). Also, the present inventorshave disclosed, in addition to these known compounds, a highly activebifidobacteria proliferation promotion substance, and have confirmedthat this substance is 2-amino-3-carboxy-1,4-naphthoquinone, which is aconventionally unknown (in other words, novel) substance, and isproduced intracellularly and extracellularly by a bacterium belonging tothe genus Propionibacterium (Japanese Patent Application Laid-Open(kokai) No. 8-98677). Furthermore, the present inventors have found that2-amino-3-carboxy-1,4-naphthoquinone is useful as a drug for preventingor treating metabolic bone diseases such as osteoporosis (WO 01/28547).

Meanwhile, DHNA has been known to be useful as an industrial materialsuch as a dye, a pigment, or a photosensitive material, and variousorganic chemical synthetic methods for DHNA have been developed (e.g.,Japanese Patent Application Laid-Open (kokai) Nos. 57-128655, 59-186942,and 60-104037). However, such a conventional synthetic method requireshigh-temperature, high-pressure reaction in an organic solvent, oremployment of, for example, an inedible reagent as a catalyst or thelike. In addition, difficulty is encountered in completely removing,from DHNA produced through such a method, solvents or reagents employedfor production thereof. Therefore, DHNA produced through such aconventional production method has not been considered to be useful infoods and beverages or in drugs.

DISCLOSURE OF THE INVENTION

The present inventors have conducted further studies on variouscompounds exhibiting a proliferation promotion effect specific tobifidobacteria, and as a result have found that a large amount of1,4-dihydroxy-2-naphthoic acid (DHNA) is produced intracellularly andextracellularly by a bacterium belonging to the genus Propionibacterium,and that a composition containing 1,4-dihydroxy-2-naphthoic acidcollected from the resultant culture broth, or 1,4-dihydroxy-2-naphthoicacid or a salt thereof, exhibits the effect of alleviating abdominalailments caused by milk intolerance, which occur upon ingestion of milk,and is useful for preventing or treating metabolic bone diseases, sincethis compound promotes differentiation of osteoblasts and expression ofosteoblast function, and suppresses formation of osteoclasts. Thepresent invention has been accomplished on the basis of these findings.Examples of 1,4-dihydroxy-2-naphthoic acid salts includepharmacologically or sitologically acceptable salts. Typical examples ofthe salts include acetates, benzenesulfonates, benzoates, bicarbonates,lactates, and citrates, which should not be construed as limiting thepresent invention thereto.

Accordingly, the present invention provides a process for producing1,4-dihydroxy-2-naphthoic acid, which comprises culturing amicroorganism capable of producing 1,4-dihydroxy-2-naphthoic acid, tothereby allow the microorganism to produce 1,4-dihydroxy-2-naphthoicacid in culture broth, and collecting the thus-produced1,4-dihydroxy-2-naphthoic acid.

The present invention also provides a composition containing1,4-dihydroxy-2-naphthoic acid, which composition is produced throughthe aforementioned production process.

The present invention also provides food and beverage for alleviatingabdominal ailments, an agent for alleviating abdominal ailments, anintestinal function regulating agent, food and beverage for preventingor treating metabolic bone diseases, or an agent for preventing ortreating metabolic bone diseases, which comprises, as an activeingredient, a composition containing 1,4-dihydroxy-2-naphthoic acid,which composition is produced through the aforementioned productionprocess, or 1,4-dihydroxy-2-naphthoic acid or a salt thereof.

The present invention also provides use of a composition containing1,4-dihydroxy-2-naphthoic acid, which composition is produced throughthe aforementioned production process, or 1,4-dihydroxy-2-naphthoic acidor a salt thereof, for producing food and beverage for alleviatingabdominal ailments, an agent for alleviating abdominal ailments, anintestinal function regulating agent, food and beverage for preventingor treating metabolic bone diseases, or an agent for preventing ortreating metabolic bone diseases.

The present invention also provides a method for treating abdominalailments, a method for regulating intestinal function, or a method fortreating metabolic bone diseases, which comprises administering, to asubject in need thereof, an effective dose of a composition containing1,4-dihydroxy-2-naphthoic acid, which composition is produced throughthe aforementioned production process, or 1,4-dihydroxy-2-naphthoic acidor a salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the intensity of abdominal distention reportedby subjects until six hours has elapsed after ingestion of a milkbeverage incorporating the composition of the present inventioncontaining a high concentration of DHNA.

FIG. 2 is a graph showing the frequency of abdominal distention reportedby subjects until six hours has elapsed after ingestion of the milkbeverage incorporating the composition of the present inventioncontaining a high concentration of DHNA.

FIG. 3 is a graph showing the frequency of tympanites (borborygmus)reported by subjects until six hours has elapsed after ingestion of themilk beverage incorporating the composition of the present inventioncontaining a high concentration of DHNA.

FIG. 4 is a graph showing the effect of DHNA on promoting calcificationof osteoblasts.

FIG. 5 is a graph showing the effect of DHNA on suppressing a decreasein bone density.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention employs a bacterium capable of producing1,4-dihydroxy-2-naphthoic acid (DHNA). Examples of the genus to whichsuch a bacterium belongs include Propionibacterium, Enterobacter,Sporolactobacillus, and Bacillus. Most of these microorganisms haveconventionally been employed for producing foods and beverages anddrugs, and therefore, employment of such a bacterium for producing aDHNA-containing food and beverage or a DHNA-containing drug ispreferred. Examples of propionic acid bacteria include bacteria forproducing cheese, such as Propionibacterium freudenreichii, P. thoenii,P. acidipropionici, and P. jensenii; P. avidum; P. acnes; P.lymphophilum; and P. granulosam. Examples of bacteria belonging to thegenus Bacillus include Bacillus subtilis and Bacillus coagulans. Themicroorganism employed in the present invention is preferablyPropionibacterium freudenreichii. Specific examples include P.freudenreichii IFO 12424 and P. freudenreichii ATCC 6207.

In order to produce DHNA through the process of the present invention,firstly, a strain capable of producing DHNA is aerobically oranaerobically cultured in a nutrient-containing medium in which generalmicroorganisms can be grown. The nutrient may be a known nutrient whichhas conventionally been employed for culturing of microorganisms. Thenutrient-containing medium is particularly preferably a mediumcontaining skim milk powder; a medium containing trypticase, phytone,yeast extract, and glucose; or a medium predominantly containinglactase-treated whey mineral, and whey powder, protease-treated wheypowder, a whey protein concentrate, or a product obtained throughtreatment of the concentrate. In the present invention, most preferably,protease-treated skim milk powder is employed as a protein source in amedium. In the case where protease-treated skim milk powder is employedin a medium, when at least one of yeast extract and lactose is employedas an additive during the course of culturing, the amount of DHNAproduced in the resultant culture broth can be increased. During thecourse of culturing, in place of lactose, glucose or lactase-treatedlactose may be employed as an additive. However, when protease-treatedskim milk powder is employed as a primary raw material for a medium,most preferably, lactose is employed as a sugar in the medium. Next willbe described an exemplary medium preparation method in the case whereprotease-treated skim milk powder is employed as a medium raw material.

Skim milk powder is dissolved in water such that the skim milk powderconcentration is 10% (w/v), followed by protein degradation by means ofprotease. The amount of protease to be employed is 0.25% (w/w) on thebasis of the entirety of the skim milk powder. Protein degradation isperformed at 47° C. and a pH of 6.8 for six hours, and an aqueouspotassium carbonate solution is employed for pH adjustment duringprotein degradation. The skim milk powder concentration (final mediumconcentration) is regulated to 10% (w/v), and finally, yeast extract isadded such that the amount of the yeast extract is 1 to 10% (w/w),preferably 3 to 7% (w/w), on the basis of the entirety of the skim milkpowder.

A DHNA-producing strain may be cultured by means of any of a variety ofknown aerobic and anaerobic culture methods, but an aerobic or anaerobicculture method employing a liquid medium is most preferred, from theviewpoint of mass production of DHNA. Culturing is performed under thefollowing conditions: incubation temperature: about 20 to about 40° C.,medium pH: neutral to slightly acidic (preferably 5.5 to 7.5). In thecase of liquid culturing, when about one to about five days elapse afterinitiation of culturing, DHNA is accumulated in the medium and thestrain. The amount of DHNA to be produced increases through addition oflactose during the course, of culturing. The thus-produced DHNA may becollected from the resultant culture broth immediately after completionof culturing. However, preferably, the culture broth is cooled (at 3 to20° C., more preferably at about 10° C.) and stored (preferably forabout two to about four weeks), to thereby allow DHNA to furtheraccumulate in the culture broth.

A DHNA collection method will next be described. Preferably, theabove-obtained culture broth is subjected to adsorption chromatography.Examples of the adsorbent which may be employed include absorbents forreversed-phase chromatography, such as activated carbon and syntheticabsorbents (e.g., Diaion HP-20, product of Mitsubishi ChemicalCorporation). Firstly, a column is filled with an absorbent, and thecolumn is washed with a 0.5% (w/v) aqueous sodium ascorbate solution.Subsequently, the above-obtained culture broth is added to the column (afluid which passes through the column is denoted by “pass”), and awater-soluble fraction is removed from the culture broth by use of a0.5% (w/v) aqueous sodium ascorbate solution. Thereafter, the resultantculture broth is subjected to elution with ethanol containing 0.5% (w/v)sodium ascorbate, and the ethanol-eluted fraction is concentrated, tothereby produce a composition containing a high concentration of DHNA.When the DHNA-containing composition is further purified, pure DHAN or asalt thereof can be produced. In place of ethanol, methanol may beemployed for eluting DHNA from the column.

Examples of DHNA salts include pharmacologically or sitologicallyacceptable salts. Typical examples of the salts include acetates,benzenesulfonates, benzoates, bicarbonates, lactates, and citrates,which should not be construed as limiting the present invention thereto.

DHNA is contained in the culture broth of the DHNA-producing bacterium(intracellularly and/or extracellularly). Therefore, rather than beingsubjected to adsorption chromatography, the culture broth per se may beconcentrated by use of, for example, a rotary evaporator, to therebyyield a composition containing a high concentration of DHNA. Preferably,the bacterium cells are separated from the culture broth by means of agenerally employed centrifugation technique, and the resultantsupernatant is concentrated. The physical form of the thus-obtainedcomposition varies in accordance with the intended use thereof; forexample, the composition may be used in liquid form or may be formedinto a powdery product.

The term “milk intolerance” refers to the case where abdominal ailmentssuch as abdominal pain, borborygmus, and diarrhea occur after ingestionof milk. Most of such abdominal ailments are associated with lactoseintolerance, which occurs as a result of ingestion of lactose containedin milk, etc. In many cases, lactose intolerance is caused by deficiencyor reduction of lactase activity in the small intestine. The compositionof the present invention, or DHNA or a salt thereof (hereinafter may bereferred to simply as “the composition, etc.”) exhibits the effect ofalleviating abdominal ailments which occur upon ingestion of milk. Inaddition, the composition, etc. exhibits the effect of promotingdifferentiation of osteoblasts and expression of osteoblast function, aswell as the effect of suppressing formation of osteoclasts, andtherefore is useful for preventing or treating metabolic bone diseasessuch as osteoporosis. The composition, etc. may assume the form of foodand beverage, or a drug. For example, through direct administration ofthe composition, etc. in the form of a drug, through direct ingestion ofthe composition, etc. in the form of a food for special use (e.g., afood for specific health use) or a nutritional food, or throughingestion of various foods (e.g., milk, fermented milk, and yoghurt)containing the composition, etc., intestinal flora can be improved,abdominal ailments which occur, for example, upon ingestion of milk canbe alleviated, and metabolic bone diseases can be prevented or treated.

In the case where the composition of the present invention, or DHNA or asalt thereof is employed as a drug, the physical form of thecomposition, etc. can be appropriately chosen in accordance with themanner of administration. Examples of the physical form include oralforms such as a tablet, a capsule, a granule, a powder, and a syrup.Such a drug product can be prepared by means of a customary methodmaking use, as a primary agent, of a known auxiliary agent which isgenerally employed in the drug preparation technical field, such as anexcipient, a binder, a disintegrating agent, a lubricant, a flavoringagent, a deodorant, a dissolution promoter, a suspending agent, or acoating agent.

When applied to a human, preferably, such a drug product is orallyadministered thereto. The effective dose of DHNA (i.e., an activeingredient) which is orally administered to a patient to be treatedvaries in accordance with the age and pathological conditions of thepatient, but in general, the daily dose of DHNA per kg body weight of ahuman is 0.03 to 3 μg, preferably 0.1 to 1 μg.

When orally administered, the composition of the present invention, orDHNA or a salt thereof attains its intended purposes; i.e., improvementof intestinal flora, alleviation of abdominal ailments which occur uponingestion of milk, and prevention or treatment of metabolic bonediseases. Therefore, the composition, etc. can be employed in the formof food and beverage. Such DHNA-containing food and beverage can beprepared by means of any of various techniques; for example, thecomposition of the present invention containing a high concentration ofDHNA, or DHNA or a salt thereof is added to any of a variety ofauxiliary agents or foods and beverages, to thereby prepare variousfoods and beverages (e.g., drink products and tablets); or thecomposition, etc. is added directly to foods and beverages. Thethus-prepared food and beverage enables DHNA to be ingested over a longperiod of time, and thus can be commercially provided in the form ofgeneral food and beverage, food for special use (e.g., food for specifichealth use), or nutritional food.

EXAMPLES

The present invention will next be described in more detail withreference to Test Example and Examples, but the present invention is notlimited to these Examples.

Test Example 1 Screening of DHNA-Producing Bacteria Culturing Conditions

Each of the below-described test bacteria was inoculated into a mediumcontaining skim milk powder (described below in Example 1), and thebacterium was anaerobically cultured by means of the GasPak method at37° C. for 18 to 72 hours.

(A) Propionibacterium freudenreichii IFO 12424 (culture time: 72 hours)

(B) Propionibacterium acidipropionicii IFO 12425 (72 hours)

(C) Propionibacterium jensenii IFO 12427 (72 hours)

(D) Lactococcus lactis ATCC 10697 (24 hours)

(E) Leuconostoc mesenteroides JCM 9700 (24 hours)

(F) Lactobacillus acidophilus ATCC 4357 (18 hours)

(G) Lactobacillus plantarum IFO 12006 (18 hours)

(H) Lactobacillus rhamnosus JCM 1136 (18 hours)

(I) Lactobacillus casei ATCC 7469 (18 hours)

(J) Bifidobacterium longum ATCC 15707 (18 hours)

(K) Bifidobacterium bifidum ATCC 11146 (18 hours)

(L) Bifidobacterium adolescentis ATCC 15703 (18 hours)

(M) Bifidobacterium breve ATCC 15700 (18 hours)

DHNA Analysis Conditions (HPLC Analysis)

Column: C18, filler particle size: 3 μm, inner diameter: 4.6 mm, length:150 mm (C18: Cadenza CD-C18, product of Imtakt Corporation)

Eluent: acetonitrile : methanol : water : acetic acid=10:20:200:0.1 (pH:7.0 (adjusted with 5% aqueous ammonia))

Flow rate: 1.5 mL/min

Injection amount: 20 μl

Detector: UV 254 nm

HPLC Sample Preparation Method

Sodium ascorbate (0.1% (w/v)) was added to the above-obtained culturebroth (10 ml), the pH of the resultant mixture was adjusted to 7.0, andwater was added to the mixture such that the total volume of theresultant mixture was 20 ml. Thereafter, an aliquot (3 ml) of themixture was mixed with methanol (3 ml), and the resultant mixture wassubjected to centrifugation at 3,000 rpm for 10 minutes. Thethus-obtained supernatant was subjected to filtration by use of a0.45-μm filter.

Quantification of DHNA

The amount of DHNA contained in the above-prepared HPLC sample wascalculated on the basis of the following previously obtained data of acommercially available DHNA standard (product of Wako Pure ChemicalIndustries, Ltd.): retention time=around 13 minutes, and relationshipbetween HPLC peak area and DHNA concentration (calibration curve).

As a result, each of the culture broths of Propionibacteria (A) through(C) was found to contain DHNA in an amount of 3.0 μg/ml or more.Although a trace amount of DHNA was detected in the culture broth ofLactococcus (D) or Leuconostoc (E), no DHNA was detected in each of theculture broths of Lactobacilli (F) through (I) and Bifidobacteria (J)through (M). That is, Propionibacterium was found to be desirable as aDHNA-producing bacterium employed in the present invention (Table 1).Meanwhile, Bacillus subtilis was aerobically cultured by use of a mediumsimilar to that described above. As a result, the thus-obtained culturebroth was found to contain DHNA.

TABLE 1 Test Bacteria Culture time (hr) DHNA (μg/ml) (A) 72 3.0 (B) 3.2(C) 3.6 (D) 24 0.1 (E) 24 0.2 (F) 18 N.D. (G) N.D. (H) N.D. (I) N.D. (J)18 N.D. (K) N.D. (L) N.D. (M) N.D. N.D.: not detected

Example 1 Production Process for DHNA-Containing Composition

Beer yeast extract (product of Asahi Breweries, Ltd.) (0.1% (w/v)) wasadded to a skim milk medium (a solution containing 10 wt. % skim milkpowder) which had been prepared by dissolving skim milk powder in watersuch that the skim milk powder concentration was 10% (w/v). Theresultant medium (50 L) was placed into 20 Erlenmeyer flasks (volume: 5L each) in a divided manner, and the medium in each of the flasks wassterilized at 121° C. for seven minutes by use of an autoclave. Anactivated culture broth of Propionibacterium freudenreichii IFO 12424strain (60 ml) was inoculated into each of the resultant media, andanaerobic culturing was performed in a nitrogen atmosphere at 37° C. for72 hours, to thereby yield a composition containing1,4-dihydroxy-2-naphthoic acid in an amount of 3 μg/ml (50 L). Theaforementioned activated culture broth was prepared by inoculatingPropionibacterium freudenreichii (2% (w/v)) into a TPYG medium(trypticase (BBL) (8 g), phytone peptone (BBL) (3 g), beer yeast extract(5 g), L-cysteine hydrochloride (0.5 g), glucose (20 g), K₂HPO₄ (2 g),KH₂PO₄ (3 g), MgCl₂.6H₂O (0.5 g), FeSO₄.7H₂O (10 mg), H₂O (1,000 ml), pH6.5), followed by anaerobic culturing by means of the GasPak method at37° C. for 72 hours.

Example 2 Production Process for Composition Containing a HighConcentration of DHNA

Skim milk powder was dissolved in water such that the skim milk powderconcentration became 10 to 20% (w/w), and protease [Amano A] (product ofAmano Pharmaceutical Co., Ltd.) was added to the resultant solution suchthat the amount of the protease was 0.25% (w/w) on the basis of theentirety of the skim milk powder, followed by enzymatic degradation ofthe resultant mixture at 47° C. for six hours. During the course ofenzymatic degradation, the pH of the mixture was maintained at 6.8 byuse of an aqueous potassium carbonate solution. The resultant reactionmixture was heated at 85° C. for five minutes to inactivate the enzyme,and then water was added to the resultant mixture such that the skimmilk powder concentration became 10% (w/w). After bear yeast extract(product of Asahi Breweries, Ltd.) was added to the resultant mixturesuch that the amount of the extract was 5% (w/w) on the basis of theentirety of the skim milk powder, the mixture (1.5 Kg) was placed into afermenter (volume: 2 L), and then sterilized at 121° C. for sevenminutes by use of an autoclave. Nitrogen gas was brought into thefermenter such that the gas flowed above the surface of the resultantmedium, the medium was stirred at 150 rpm, and the temperature of themedium was regulated to 33° C. After the medium temperature wasstabilized at 33° C., a frozen concentrated starter of Propionibacteriumfreudenreichii ET-3 strain (deposited as FERM BP-8115 with InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology (Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki,Japan (postal code: 305-8566)), on Aug. 9, 2001) (0.05% (w/w)) wasinoculated into the medium, and anaerobic culturing was initiated.Lactose (2% (w/w)) and lactose (1.3% (w/w)) were added to the culturebroth 72 hours and 96 hours after initiation of this culturing,respectively. This anaerobic culturing was performed in an nitrogenatmosphere at 33° C. for 120 hours while the pH of the culture broth wasmaintained at 6.45 by use of a 40% (w/w) aqueous potassium carbonatesolution, and as a result, DHNA (30 μg/ml) was produced in the culturebroth. After the 120-hour culturing, the amount of the alkali consumedin the culture broth (1.5 kg) was found to be 131 g. Sodium ascorbatewas added to the resultant culture broth such that the amount of sodiumascorbate was 0.5% (w/w) on the basis of the entirety of the culturebroth, and the pH of the resultant mixture was adjusted to 8.0 by use ofan aqueous potassium carbonate solution, followed by cooling to 10° C.The resultant culture broth was stored at 10° C. for two weeks, and as aresult, the DHNA content of the culture broth was increased to 40 μg/ml.The above-employed frozen concentrated starter was prepared through thefollowing procedure: an activated culture broth of Propionibacteriumfreudenreichii ET-3 strain (prepared through anaerobic culturing of thestrain in a medium predominantly containing the aforementionedprotease-treated skim milk powder at 33° C. for 48 hours) (2% (w/w)) wasinoculated into a medium predominantly containing the protease-treatedskim milk powder, followed by culturing at 33° C. for 72 hours; aftercompletion of culturing, the culture broth was collected and thensubjected to centrifugation, to thereby increase the bacterium cellconcentration by a factor of about 20; and an appropriate amount of theresultant product was placed in a sterilized container, frozen at −80°C. or lower, and then stored at −80° C.

Example 3 Concentration of the Composition Obtained in Example 1 byMeans of Column Chromatography

A column was filled with Diaion HP-20 (4 L), and the column was washedwith a 0.5% (w/v) aqueous sodium ascorbate solution. Thereafter, thecomposition obtained in Example 1 (40 Kg) was added to the column.Subsequently, a water-soluble fraction was removed from the compositionby use of a 0.5% (w/v) aqueous sodium ascorbate solution (8 L).Thereafter, an ethanol eluent (12 L) containing 0.5% (w/v) sodiumascorbate was added to the column, to thereby elute DHNA. Theethanol-eluted fraction was concentrated by use of an evaporator, tothereby produce the composition of the present invention (10 g)containing DHNA (115 mg).

Example 4 Concentration of the Composition Obtained in Example 1 by useof a Rotary Evaporator

Sodium ascorbate (0.5% (w/w)) was added to the composition obtained inExample 1 (5 Kg), and the resultant mixture was concentrated by a factorof five by use of a rotary evaporator, to thereby produce thecomposition of the present invention (1 Kg) containing DHNA (15 mg).

Since each of the compositions of Examples 1 through 3 is produced byuse of a propionic acid bacterium, which is employed for producingcheese, the invention compositions obtained in Examples 1 through 3 canbe employed in foods and beverages, without being subjected to anytreatment.

Example 5 Purification of DHNA

The concentrate (culture broth) obtained in Example 2 was dissolved in a0.5% (w/v) aqueous sodium ascorbate solution (1 L) whose pH was adjustedto 4.5, and the resultant solution was subjected to extraction withethyl acetate (1 L) three times. The thus-obtained ethyl acetate layerswere mixed together, and then subjected to dehydration with anhydroussodium sulfate (200 g), followed by concentration under reducedpressure. After the resultant concentrate was dissolved in methanol (80mL), and an aliquot (4 mL) of the resultant solution was purified by useof a C18 column. Sodium ascorbate was added to the resultant DHNAelution fraction (retention time: 21 to 31 minutes) such that the sodiumascorbate content was 25% (w/v), and then the resultant mixture wasconcentrated under reduced pressure. The resultant concentrate (800 mL)was subjected to extraction with ethyl acetate (300 mL) twice, and thensubjected to dehydration with anhydrous sodium sulfate (50 g), followedby concentration under reduced pressure. The final purified product wasidentified as DHNA as a result of structural analysis by means of500-MHz ¹H-NMR spectroscopy. Through the above procedure, DHNA (115 mg)was produced from the culture broth (40 L).

Column: Capcell Pak C18 SG120, φ50×500 mm, Lot. 930210 (product ofShiseido Co., Ltd.)Mobile phase: acetonitrile:methanol:water:acetic acid=20:40:200:0.1 (pH:7.0 (adjusted with 5% aqueous ammonia))Temperature: room temperatureFlow rate: 100 mL/minInjection amount: 4 mL

Detector: UV 254 nm <NMR Data of the Final Purified Product>

¹H-NMR (500 MHz, MeOH-d₄): δ 8.39 (1H, d, J=8.3 Hz), 8.23 (1H, d, J=8.3Hz), 7.69 (1H, dd, J=8.3, 6.9 Hz), 7.60 (1H, dd, J=8.3, 6.9 Hz), 7.23(1H, s)

Example 6 Acute Toxicity Test

The DHNA-containing composition described in Example 2 was subjected toan acute toxicity test by use of five mice (five weeks old: commerciallyavailable ICR mice, habituated for seven days). The composition wasadministered to each of the mice for five consecutive days (maximumdaily dose: 78.3 mg/kg (DHNA: 0.9 (‥115×(78.3/(10×1000))) mg/kg)), andthe thus-administered mice were observed for 14 days. As a result, noneof the mice died, and none of the mice were confirmed to exhibitabnormality in terms of body weight, behavior, and dissected organs.

Example 7 Preparation Method for Food Incorporating the DHNA-ContainingComposition of the Present Invention (Tablet Preparation)

The composition obtained in Example 1 (10 Kg) was freeze-dried at atemperature of 50° C. for 24 hours, to thereby yield freeze-dried powder(1 Kg). Subsequently, the powder (40% (w/w)) was incorporated into atablet base material containing glucose (80% (w/w)), dry corn starch(10% (w/w)), palatinit powder (7% (w/w)), and citric acid (3% (w/w)),and the resultant material was tableted each tablet weighing 0.5 g.

Example 8 Preparation Method for Food Incorporating the DHNA-ContainingComposition of the Present Invention (Milk Beverage Preparation 1)

Sodium ascorbate (15 g) and the DHNA-containing composition obtained inExample 2 (125 mg) were added to raw milk (10 Kg), and the resultantmixture was homogenized and then sterilized at 130° C. for two seconds.Every 100 ml of the resultant product was charged into a container.

Example 9 Preparation Method for Food Incorporating the DHNA-ContainingComposition of the Present Invention (Milk Beverage Preparation 2)

An activated culture broth of Propionibacterium freudenreichii ET-3strain (FERM P-18454) (60 ml) was inoculated into a medium predominantlycontaining protease-treated whey powder (the medium was prepared throughthe following procedure: beer yeast extract (product of Asahi Breweries,Ltd.) (0.1% (w/v)) was added to a solution obtained through two-hourprotein degradation (50° C., pH 7.0) of a solution containing 10 wt. %whey powder by use of protease (Amano A, product of Amano PharmaceuticalCo., Ltd.), and the resultant mixture (50 L) was placed in a jarfermenter and then sterilized at 121° C. for seven minutes), followed byanaerobic culturing at 35° C. and a pH of 6.0 for 90 hours, and sodiumascorbate (0.5% (w/v)) was added to the resultant culture broth, tothereby yield the composition of the present invention. Thethus-obtained composition (177.5 ml) was added to raw milk (9822.5 ml),and the resultant mixture was homogenized and then sterilized at 130° C.for two seconds. Every 100 ml of the resultant product was charged intoa container (DHNA content: 11 μg/100 ml).

The aforementioned activated culture broth was prepared by means of thesame process as described in Example 1, except that the incubationtemperature was changed to 35° C.

Example 10 Effect of the Invention Composition on Abdominal Ailmentsupon Milk Beverage Ingestion 1

The milk beverage prepared in Example 8 was employed as a test beverage,and a milk beverage which had been prepared in a manner similar to thatof Example 8 by use of a non-fermented medium was employed as a controlbeverage. The subjects of the test were selected from among humans whoexhibited low β-galactosidase activity in the small intestine when theywere subjected to measurement of breath hydrogen concentration uponingestion of milk, and thus were considered to suffer from lactoseintolerance. Specifically, the subjects were 15 humans (seven males andeight females, mean age: 28.07±3.41) who underwent a lactose load testby use of the control beverage (400 ml), and exhibited an increase inbreath hydrogen concentration by about 20 ppm or more within six hoursafter ingestion of the beverage.

The subjects were instructed to abstain from ingesting any food orbeverage (except for water) from 22:00 on the day prior to testing to17:00 on the day of testing. At 10:00 on the day of testing, the controlbeverage (400 ml) was orally administered to each of the subjects. Until17:00 of the day, a questionnaire on abdominal conditions was filled outby the subject (every 30 minutes), and the breath was sampled from thesubject (every hour). One week later, the test beverage was administeredto each of the subjects in a manner similar to the control beverageadministration. The subjects were not previously informed of the typesof these beverages.

The breath of each of the subjects was sampled by use of a Tedlar baghaving a cock (volume: 1 L) (GL Sciences, Inc.), and hydrogen gasanalysis was performed by use of a gas chromatograph (GC-8A, ShimadzuCorporation) under the following analysis conditions: column: molecularsieve 5A (3 mm×2 m), oven temperature: 40° C., carrier gas: argon,detector: TCD (thermal conductivity detector).

A questionnaire on abdominal conditions was handed out to each of thesubjects, and was filled out by the subject every 30 minutes. Abdominaldistention was rated with numerical points in accordance with thefollowing criteria:

4: significant abdominal flatulence as compared with the stateimmediately after ingestion,

3: moderate abdominal flatulence as compared with the state immediatelyafter ingestion,

2: slight abdominal flatulence as compared with the state immediatelyafter ingestion, and

1: no perceivable change as compared with the state immediately afteringestion. These numerical ratings as obtained at 30-minute intervals(from 30 minutes to six hours) after ingestion were accumulated. Anyother abdominal conditions, including diarrhea, abdominal pain, andtympanites (borborygmus), were also described in the questionnaire.

The evaluation results regarding abdominal conditions from the timeimmediately after ingestion of the milk beverages to six hours afteringestion thereof are as follows. In the case of ingestion of thecontrol beverage, the cumulative value of the abdominal distention scorewas found to be 17.93±4.83. In contrast, in the case of ingestion of thetest beverage, the cumulative value was found to be 15.93±3.65, which issignificantly lower than the value obtained in the case of the controlbeverage (Wilcoxon test, p<0.05) (FIG. 1). In the case of ingestion ofthe control beverage, the frequency of abdominal distention reported bythe subjects until six hours elapsed after ingestion was found to be3.47±3.11. In contrast, in the case of ingestion of the test beverage,the abdominal distention frequency was found to be 2.47±2.90, which islower than the abdominal distention frequency obtained in the case ofingestion of the control beverage (p=0.108) (FIG. 2). In the case ofingestion of the control beverage, the frequency of tympanites(borborygmus) reported by the subjects until six hours elapsed afteringestion was found to be 2.87±2.75. In contrast, in the case ofingestion of the test beverage, the tympanites frequency was found to be1.47±2.10, which is significantly lower than the tympanites frequencyobtained in the case of ingestion of the control beverage (p<0.05) (FIG.3). Meanwhile, in the case of ingestion of the control beverage, twosubjects complained of diarrhea, but, in the case of ingestion of thetest beverage, no subjects complained of diarrhea. In the case ofingestion of the control beverage, the number of subjects who did notsuffer from abdominal ailments was two, but, in the case of ingestion ofthe test beverage, the number of such subjects was increased to six(Table 2).

In the case of ingestion of the control beverage, the average value ofmaximum increases in breath hydrogen concentration was found to be42.9±13.7 ppm. In contrast, in the case of ingestion of the testbeverage, the average value of maximum increases in breath hydrogenconcentration was found to be 34.7±17.6 ppm, which is lower than theaverage value obtained in the case of ingestion of the control beverage(Wilcoxon test, p=0.051) (Table 2).

TABLE 2 Effects of ingestion of milk beverage incorporating theDHNA-containing composition on abdominal ailments and breath hydrogenconcentration Ingestion of control beverage Ingestion of test beverageSubjects Breath hydrogen Abdominal Breath hydrogen Abdominal No. Age Sex concentration* condiitions^(§)  concentration* conditions^(§) 1 27Female 30.2 B, C 21.8 B 2 25 Female 50.0 N 33.9 N 3 28 Female 19.2 B, C 6.7 C 4 24 Female 32.1 B, C 13.6 B 5 26 Male N.T. C N.T. C 6 32 MaleN.T. C N.T. N 7 28 Male N.T. C N.T. C 8 27 Female 54.8 A, B, C 46.6 B, C9 27 Male 51.0 C 44.2 N 10 32 Male 41.2 N 21.5 N 11 25 Female 42.2 B38.1 B, C 12 23 Female 30.8 B 59.1 N 13 34 Female N.T. A, C N.T. N 14 33Male 63.2 C 60.0 C 15 30 Male 57.7 C 35.7 C Mean ± S.D. 42.9 ± 13.7 34.7± 17.6 *Maximum increases in breath hydrogen concentration (ppm) ^(§)A:Diarrhea, B: Abdominal pain, C: Tympanites, N: No subjective symptomN.T.: Not tested.

Example 11 Effect of the Invention Composition on Abdominal Ailmentsupon Milk Beverage Ingestion 2

Culturing of Propionibacterium freudenreichii IFO 12424 strain wasperformed in a manner similar to that in Example 9. The thus-obtainedculture broth (5 Kg) was concentrated by a factor of five by use of anevaporator, to thereby yield a composition (1 Kg) containing DHNA (45mg). A milk beverage was prepared from the thus-obtained composition(35.5 g), raw milk (10 kg), and sodium ascorbate (15 g), and theresultant milk beverage was subjected to testing in a manner similar tothat in Example 10. In the case of ingestion of the milk beveragecontaining the composition of the present invention, effectssubstantially similar to those obtained in Example 10 were obtained, andabdominal ailments upon milk beverage ingestion were found to bealleviated.

Example 12 Effect of DHNA on Promoting Calcification of Osteoblasts

Human osteoblasts (SaM-1), which had been cultured from long boneperiosteum of a 20-year-old male obtained during fracture surgery, wereemployed. The SaM-1 cells exhibited all the characteristic features ofosteoblasts (Koshihara, Y. et al.: In Vitro Cell. Dev. Biol., 25: 37-43,1989). SaM-1 has been known to promote calcification in the presence of2 mM of α-glycerophosphoric acid in a 1 α, 25 (OH)₂D₃concentration-dependent manner (Koshihara, Y. et al.,: Biochem. Biophys.Res. commun., 145: 651-657, 1987).

The above-obtained SaM-1 cells of 18 PDL (population doubling level)were inoculated into a 12-well plate, and cultured until the cellsreached a confluent state. Subsequently, α-glycerophosphoric acid, whichis a calcification promoting agent, was added to the resultant culturebroth such that the α-glycerophosphoric acid content became 2 mM. DHNA(10⁻⁷ M to 10⁻⁵ M) was added to the culture broth, followed by culturingfor 32 days. For a control, DMSO (a solvent) was added to the culturebroth such that the DMSO content became 0.1%. The media of the testculture broth and the control culture broth were exchanged every secondday with a DHNA-containing medium and a DMSO-containing medium,respectively. The degree of calcification was represented by the amountof Ca, which constitutes hydroxyapatite.

The Ca content of extracellular matrix was quantified by use of a kit(Calcium C Test Wako) based on the o-cresolphthalein complexone method(OCPC method) (Gitleman, H. J.: Anal. Biochem., 18: 520-531, 1967).

After completion of culturing, the resultant cells were washed with aHanks' solution. Cold 5% perchloric acid (0.5 ml/well) was added to thecells, followed by shaking extraction at 4° C. for 15 minutes. Theresultant extract (25 μL) was mixed with a buffer (2.5 mL), andsubsequently a coloring solution (containing OCPC (0.4 mg/mL) and8-quinolinol) (250 was added to the resultant mixture and then stirredfor five minutes. Thereafter, the resultant reaction mixture wassubjected to measurement by use of an absorbance meter (570 nm) (FIG.4). As shown in FIG. 4, DHNA promotes calcification in aconcentration-dependent manner.

Example 13 Effect of DHNA on Animal Model with Osteoporosis Induced byFK-506

As has been known, administration of FK-506, which is known as animmunosuppressive agent, to an animal induces osteoporosis-likeconditions (J. Hard Tissue Biology, 103-107, 10 (2), 2001). As has beensuggested, formation of osteoclasts proceeds by means of hyperexpressionof RANKL (osteoclast differentiation factor), which is expressed onosteoblasts, and bone resorption exceeds bone formation, resulting insuch osteoporosis-like conditions. FK-506 (1 mg/kg) wasintraperitoneally administered to ICR male mice (eight weeks old) for 10consecutive weeks. During the 10-week FK-506 administration, a feed(CRF-1, product of Oriental Yeast Co., Ltd.) was arbitrarily ingestedinto the mice, and DHNA (75 μg/kg) which had been suspended in a 1%aqueous DMSO (dimethyl sulfoxide) solution was orally administered toeach of the mice every day. As a result, the bone density of the mice inthe DHNA administration group was found to be significantly higher thanthat of the mice in the control group (FK 506 (+)), and a decrease inbone density due to FK 506 administration was found to be suppressed bymeans of DHNA (FIG. 5).

INDUSTRIAL APPLICABILITY

A DHNA-containing composition produced through the industrial productionprocess for DHNA of the present invention is derived from amicroorganism, and thus the composition exhibits excellent safety. Whenorally administered, the composition containing a high concentration ofDHNA improves intestinal flora. In addition, the composition can beemployed for alleviating abdominal ailments which occur upon ingestionof milk, and for preventing or treating metabolic bone diseases. Byvirtue of its non toxicity, the composition can be ingested over a longperiod of time.

1.-22. (canceled)
 23. A method for regulating intestinal functioncomprising: administering to a subject in need thereof an effective doseof 1,4-dihydroxy-2-naphthoic acid or a salt thereof.
 24. The method ofclaim 23, wherein said subject has an abdominal ailment.
 25. The methodof claim 23, wherein said subject is intolerant to milk or intolerant tolactose.
 26. The method of claim 23, wherein said subject has at leastone of abdominal pain, borborygmus, or diarrhea after ingestion of milk.27. The method of claim 23, comprising a administering an isolated orpurified 1,4-dihydroxy-2-naphthoic acid or a salt thereof.
 28. Themethod of claim 23, wherein said effective dose comprises apharmacologically or sitologically acceptable salt of1,4-dihydroxy-2-naphthoic acid.
 29. The method of claim 23, wherein saideffective dose comprises at least one salt of 1,4-dihydroxy-2-naphthoicacid selected from the group consisting of an acetate, abenzenesulfonate, a benzoate, a bicarbonate, a lactate and a citratesalt.
 30. The method of claim 23, comprising administering an effectivedose of 1,4-dihydroxy-2-naphthoic acid or a salt thereof that has beenincorporated into a nutritional food or beverage.
 31. The method ofclaim 23, comprising administering an effective dose of1,4-dihydroxy-2-naphthoic acid or a salt thereof that has beenincorporated into milk, fermented milk, yoghurt, or cheese.
 32. Themethod of claim 23, wherein said effective dose further comprises anauxiliary agent selected from the group consisting of an excipient, abinder, a disintegrating agent, a lubricant, a flavoring agent, adeodorant, a dissolution promoter, a suspending agent and a coatingagent.
 33. The method of claim 23, wherein said effective dose is in aform suitable for oral administration.
 34. The method of claim 23,wherein said effective dose is in a form of a drug.
 35. The method ofclaim 23, wherein said effective dose is in a form of a compositionselected from the group consisting of a tablet, a capsule, a granule, apowder and a syrup.
 36. The method of claim 23, wherein said effectivedose comprises a composition containing 1,4-dihydroxy-2-naphthoic acidor a salt thereof made by culturing a microorganism that produces1,4-dihydroxy-2-naphthoic acid or a salt thereof in a culture medium fora time and under conditions suitable for production of1,4-dihydroxy-2-naphthoic acid or a salt thereof.
 37. The method ofclaim 36, wherein said effective dose comprises: culture mediumcontaining 1,4-dihydroxy-2-naphthoic acid or a salt thereof and amicroorganism of the genus Propionibacterium.
 38. The method of claim36, wherein the microorganism is selected from the group consisting ofP. freudenreichii, P. thoenii, P. acidipropionici, P. jensenii, P.avidum, P. acnes, P. lyphophilum and P. granulosam.
 39. The method ofclaim 36, wherein the microorganism is P. freudenreichii.
 40. The methodof claim 36, wherein the microorganism is selected from the groupconsisting of P. freudenreichii IFO 12424, P. freudenreichii ATCC 6207,P. freudenreichii ET-3 FERM BP-8115 and P. freudenreichii ET-3 FERMP-18454.
 41. The method of claim 36, wherein said composition comprisesmore than 3.6 μg/mL of 1,4-dihydroxy-2-naphthoic acid or a salt thereof.42. The method of claim 36, wherein said composition comprises a culturebroth containing a microorganism of the genus Propionibacterium and1,4-dihydroxy-2-naphthoic acid or a salt thereof, which has beenproduced by a process comprising: culturing the microorganism of thegenus Propionibacterium in a culture medium comprising a protein sourceto thereby produce the culture broth comprising the microorganism of thegenus Propionibacterium and 1,4-dihydroxy-2-naphthoic acid or a saltthereof; storing the culture broth for a period of 2-4 weeks at atemperature of 3-20° C.; and collecting from the culture medium theculture broth comprising the microorganism of the genusPropionibacterium and 1,4-dihydroxy-2-naphthoic acid or a salt thereof.43. The method of claim 42, wherein said process further comprises,after said collecting: concentrating the culture broth.
 44. The methodof claim 42, wherein the microorganism is selected from the groupconsisting of P. freudenreichii, P. thoenii, P. acidipropionici, P.jensenii, P. avidum, P. acnes, P. lyphophilum and P. granulosam.
 45. Themethod of claim 42, wherein the microorganism is P. freudenreichii. 46.The method of claim 42, wherein the microorganism is selected from fromthe group consisting of P. freudenreichii IFO 12424, P. freudenreichiiATCC 6207, P. freudenreichii ET-3 FERM BP-8115 and P. freudenreichiiET-3 FERM P-18454.
 47. The method of claim 42, wherein the culture brothcomprises more than 3.6 μg/mL of 1,4-dihydroxy-2-naphthoic acid or asalt thereof.